Vitamin b12 factor



May 17, 1960 K. BERNHAUER ETAL 2,937,167

VITAMIN Bz FACTOR Filed July 15. 1957 3 sheets-sheet 1 'QT' up u 1 e v Q. t 1 ,i .i Q R g; x Y1 Q d Q K I q u q Ka 1 a q w Q E l l l 5 E l s Tons M'". l) Y #zum www WMM May 17, 1960 K. BERNHAUER ErAL 2,937,167

l VITAMIN Blz FACTOR Filed July 15, 1957 s sheets-sheet:

BY WMM- y.- WW

May 17, 1960 K. BERNHAUER ETAL 2,937,167

VITAMIN B.l FACTOR Filed July 15. 1957 I5 Sheets-Sheet 3 HZ- oo NH2 @H2 CHa CU CH2- oo NH2 4 NH2' CO CH2 feo-CH2l cHg" CH3 NH HZ N N ol 0H c/ H0H/H HOCH2 IN VEN TORS XMJW BY Mddw .um

for

United States Patent fenburg am Main, Germany, assignors, by mesne asi signments, to Merck & Co., Inc., Rahway, NJ., a corporation of New Jersey I Application July 15, 1957, Serial No. 671,880 Claims priority, application Germany August 14, 1953 1 Claim. (Cl. 2641-2115) The present invention relates to the production and isolation of a therapeutically valuable substance and more particularly -to a new vitamin B12 factor and also to the method of separating and differentiating this new vitamin B12 factor from other similar substances. Y

This application is -a continuation-impart of our copending application Serial No. 449,318, filed August' 12, 1954, for New Vitamin B12 Factor, now abandoned.

Itis an object of the present invention to provide a new composition of matter whichv shall herein always be designated as vitamin B12 factor III. This designation is given because of the similarity and relation of the composition of the present invention to the other factors of vitamin B12 and adequate directions for the isolation and identification of the vitamin vB12 factor III of the present invention will be given herein.

It is another object of the present invention'to provide a methodv of isolating vitamin B12 factor lll from a digested sludge.

It is yet another object of the present invention to pro-A vide a method of obtaining crystalline vitaminl B12 factor III.

It is a further object o f the present invention to provide a method for the chromatographic separation of vitamin B12 factor III from the other vitamin B12 factors.

Other objects and advantages ofthe present invention will be apparent from a further reading of the-specification and the appended claim.

The new chemical compound to which this invention refers is provisionally designated as vitamin VB12 factor III. It differs from allv similar compounds hitherto known Iand lalso from the hitherto known vitamin B12 itself. It is able to permit the growth of the E. coli mutant 1l3-3, of Lactobacillus lechmanni 313, and of Ochromonas malhamenss (Pringsheim), and has a striking and eiective action in the therapeutic treatment of pernicious anemia and other macrocytic anemias.

The compound of the present invention was found to have the structural formula shown in Fig. 3 of the drawing. It will be noted that the compound of the present invention differs from vitamin B12 itself in that while vitamin B12 has a methyl group in each of the 5- and -positions of the benzimidizole portion ofthe compound, the factor III of the present invention has a hydroxyl group in the 5-position and no substituent in the 6-position.

Vitamin B12 factor III of the present invention has been 'found to bev highly effective as an antipernicious anemia factor andthis has been'proved by clinical tests such as those vdescribed in Arzneimittel-Forschung 5,442-446 (1955).

The digested sludge obtainable in the anaerobic digestion of sewage sludge by methane fermentation can be used as the starting material for producing this substance. An aqueous extract is first produced therefrom, and from said extract a concentrate which contains, in addition to other representatives of the vitamin B12 group and other impurities, the new vitamin B12 factor III. The crystal- 2,937,167 Patented May 17, 1960 iCC 2 lized vitamin is obtained by further purification of the concentrate.

The vitamin B12 factor III content in digested sludges of different origins varies considerably.' There are digested sludges in which this factor predominates and the other vitamin B12 factors, .such as for example factor B, cy-anocobalamin, pseudo-vitamin B12 aud'dfactor A, occur only in relatively small amounts. Such digested sludges contain about 0.3 mg. per litre of vitamin B12 factorV III. in approximately the same Iamount as vitamin B12, and in others again it occurs only in smaller amounts. However, it has been possible to obtain vitamin B12 factor III from almost all digested sludges.

For the purpose of producing vitamin B12 factor III `from digested sludge, the latter is mixed in the condition in which it is available or', if necessary, after dilution with a little Water, with a small amount, for example 0.1%, of a water-soluble cyanide or bisulphite or free hydrocyanic yacid or SO2, adjusted to a pH of 5-7, and heated for a short time, for example 20 to 60 minutes, to 70el20 C. The vitamin B12 factor III is thereby released from the linksto proteins or peptides and rendered extractable.

The digested vsludge thus activated is now centrifuged or filtered for the purpose of separating the solid com,- ponents. It is often advisable, in order to obtain satsfactory flocculation of the impurities to `add a small amount, for example 0.2 to 0.5% of iron (ferrie) chloride or aluminum sulphate. The clear filtrate, if it has a temperature of over 215, is cooled and for the purpose of recovering the `vitamin B12 factor `lll is stirred up with a suitable adsorption medium, such as for example activated charcoal, montmorillonite, and the like. A column 1 may also be filled with a suitable adsorbing medium and the liquid to be treated passed through the column for the purpose of -adsorption ofthe vitamin B12 factor III.

After separation of the liquid, the adsorbate is washed with water and thereupon subjected to an elution process,

whereby the vitamin B12 `factor III passes over again into a liquid phase. Elution can be carried out by stirring the adsorbate, for example activated charcoal, with a suitable elution medium, heating the same, and separating it from the elution medium. The vitamin B12 factor III is thereby concentrated in the elution medium. Elution can of course also be carried out in a column.

When using activated charcoal, aqueous ketones and aqueous alcohols are suitable for example as elution medium, e.g. aV mixture of 30 parts of water and 70 parts of ethanol, or parts` of water and 50l parts of 'isopropanoh or 90 parts of water and 10 parts of nbutanol. The eluate is concentrated in vacuo to a small volume in order to reduce the volume and eliminate the organic solvents. The resulting eluate concentrate contains the vitamin B12 factor III already in a considerable Y concentratlon, thus for example in a concentration of 20 to 8O mg. per litre. f

For the purpose of further purification of the eluate concentrates produced in the above-described manner or In other digested Ysludges this factor occurs can however also be mixed with the necessary amount of a water binding medium, such as dehydrated sodium sulphate, and thereupon ground. A dry powder is thereby. produced which can then be subjected toa percolation process.

Dry methanol or methanol having a low water content (for example containing water), or else a mixture of a phenol and a hydrocarbon, or a holagenated hydrocarbon, such as o-dichloro benzene or trichloroethylene or chloroform with 5 to 20% of p-chlorophenol or 7 to 25% of phenol, etc., or carbon disulphide with a phenol, are suitable for example as percolation liquid. During the percolation, the vitamin B12 factor III, together with other vitamins of the B12 group, is washed out of the column, while most of the impurities remain in the column. This process can also be carried out by stirring up the dry powder with one of the liquids mentioned and separating by means of a centrifuge or a filter.

` According to another method, the eluate concentrate is mixed with a suitable salt, for example with 30% of ammonium sulphate, and extracted with a low alcohol or ketone, such as isopropanol or methylethylketone, whereby the vitamin B12 factor III passes over into the organic phase. The extracts produced in this manner can be freed in vacuo from alcohol or ketone and the residue subjected to further purification. The extracts can, however, also be mixed with a suitable hydrocarbon, or better still with a suitable halogenated hydrocarbon, such as trichloroethylene, and shaken out with water, whereby the vitamin B12 factor lli passes over into the aqueous phase. After separation of this phase and removal of theorganic solvents still dissolved therein in vacuo, this aqueous extract can be subjected to a further purification process. l

By another method the eluate concentrate, or any other similar aqueous concentrate, isfsubjected to a liquid-liquid extraction, the extraction medium used comprising mixtures of hydrocarbons or halogenated hydrocarbons or carbon disulphide with halogenated phenol. The hydrocarbons or halogenated hydrocarbons used may be for example benzene, toluene, chloroform, trichloroethylene, chlorobenzene, o-dichlorobenzene. The halogenated phenols used may for example be m-chlorophenol, pchlorophenol, halogenated cresols etc. An extraction liquid which is used may, for example, contain l3-25% of p-chlorophenol or 9-20% of m-chlorophenol or 17- 28% of 6-chloro-3-cresol, etc.

This extraction can be carried out in separatory funnels, for example in the form of a counter-current distribution, or in an extraction centrifuge. rThe organic extracts containing the vitamin B12 factor IH are washed for the purpose of removing impurities, for example with a dilute solution of sodium bicarbonate or with a phosphate buffer solution of 1 5 strength and of a pH of 7-9, andthen with water. The washing water is reextracted with' a small amount of extraction medium containing phenol.

For the purpose of further purification of the red colo-red phenol-containing mixtures which contain the vitamin B12 factor IIL said mixtures are mixed with a small amount, for example 5-l5%, of a polar oxygencontaining liquid,- such as an alcohol or a ketone, ether, etc., and extracted with water. Through the addition of the polar, oxygen-containing liquid, the distribution coetlicient of the vitamin B12 factor Ill is displaced to such an extent in favor of water that the vitamin can at once be extracted with small volumes of water. Similarly to the preceding purification step, this process entails a substantial concentration and purification of vitamin B12 factor lll.

f The aqueous extracts produced by the last-described process. andcontaining the vitamin B12 factor III and other B12-like factors are thereupon freed in vacuo from the residues of organic solvents, after which they have a pure red color. Further purification of these extracts can be carried out successfully only by chromatographic processes. For this purpose, the extracts can be concentrated to dryness in vacuo and thereupon passed to the chromatographic column. It is, however, far more convenient and far less injuriousto the vitamin, as well as time-saving, to adopt the following method.

The red colored aqueous extract freed from extraneous solvents is mixed with a small amount, for example 0.5 to 2% Vof a powdered porous substance, such as kieselguhr, cellulose powder, etc., adjusted to a pH of 2.0 to 4.5, but preferably to a pH of 3.0, and vigorously shaken with a small amount of a phenolic substance, for example 8.0% of phenol or 2.2% of p-chlorophenol or 1.7% of to said phenols. These complexes are difficulty soluble in water and have an oily consistency. When shaken up with a small amount of for example kieselguhr, the oily complex containing the vitamin B12 factor III and other B12-like factors is absorbed by kieselguhr. Y The kieselguhr powder charged in this manner, which is then deep red in color, rapidly settles at the bottom. This deposit can be separated with the aid of a centrifuge or suction filter, while it is advisable to use, for example, kieselguhr as a filter aid. The red deposit sucked off is thereupon mixed with the necessary amount of acetone, or better still, of a mixture of acetone and ether, for the purpose of removing the phenolic substance, whereby the latter is dissolved and the vitamin remains on the filter (for example kieselguhr). washed with, for example, acetone, and freed from solvent. The resulting preparation, which is a pure red in color, contains the vitamin B12 factor III and vother B12- like compounds in a highly puried condition and can be directly chromatographed for the purpose of further purification.

Since the vitamin B12 factor III is always accompanied in the digested sludge byta number of other vitamin B12 factors, chromatography is a critical and decisive purification step and must be carried yout with particular care. An important prerequisite for success of the chromatographic purification is the maintenance of a defined molecular structure of all vitamin B12 factors during this operation. This condition is best fulfilled by first treating the vitamin B12 mixture to be chromatographed with, for example, gaseous hydrocyanic acid, whereby -the vitamin B12 factors are converted into cyano complexes. In addition, care must be taken that the filling materials of the chromatography column and of the developers are supplied with -a corresponding amount of cyanide ions. The vitamin B12 mixture can however also be chromatographed in the form of thiocyanate complexes; in this case the column and thedeveloper must contain the corresponding anions, such as thiocyanate, etc.

The chromatographic separation of the vitamin B12 factor III from other factors can be carried out by charging the above-described kieselguhr or cellulose preparation, after suitable treatment with, for example, hydrocyanic acid gas, into a chromatographing column prepared with aluminum oxide and acetone, and chromatographing it with a mixture of acetone and Water containing, for example, cyanide ions. The developer has to contain a 1/5000 to 1,4300 molar cyanide concentration. Development is first eected with an acetone-water mixture containing 540% of water, and finally with an acetone-water mixture containing 20 to 30% of water. The violet'colored factor B contained in every digested sludge leaves the column first,

The deposit free from phenol is now separated,V

followed by vitamin B12 and then the vitamin B12 factor III of the present invention. Other red-colored factors, if present, then follow; After concentration of the fraction containing the vitamin B12 factor III invacuo, this factor can be crystallized.

The chromatographic separation of the vitamin B12 factor III from the other vitamin B12 factors can be carried out still more successfully if a cellulose column and a mixture of n-butanol and water containing cyanide ions, for example, as developer are used. For the purpose of preparing the column, 50 g. of cellulose powder may be mixed with 500 cc. of n-butanol saturated with water. This mixture should contain at least 0.0025%, for example, of cyanide ions. 20 cc. of water, for example, are then added with vigorous shaking or stirring. It is shaken or stirred until the material is completely homogeneous. After standing for several hours, the cellulose suspension is ready for use. When using cellulose flakes instead of cellulose powder, correspondingly more water-saturated n-butanol must be used, because otherwise the mass will be too thick.

The cellulose suspension prepared `in the manner described is then filled in several portions into the chromatographing tube and compressed with the aid of a suitable tamper. The column is then washed with a corresponding amount of developer, consistingforzexample of watersaturated n-butanol, containing 0.0025 to 0.01% of for example cyanide ions, after which the material to be chromatographed, for example the kieselguhr dry preparation containing the vitamin B12 factor III together with other vitaminB12 factors, is introduced. The vitamin B12 factor III can also be introduced into the column in the form of a liquid concentrate, for example dissolved in Water-saturated n-butanol. If the material was supplied as a dry preparation, it is moistened with a little watersaturated n-butanol, and after laying on a filter paper disc it is lightly compressed with the aid of a tamper. By the addition of small amounts of developer, the vitamin B12 factors are flushed into the cellulose layer, whereupon the development can begin.

In numerous chromatograms the following R values characteristic of each factor were observed:

Vitamin B12 factor: R values Factor B e GAO-0.60 Vitamin'Blg A Factor III 0.080.12

The fluctuations of the R values are due to the different methods of production of the columns, because it is not possible to carry out this process absolutely identically each time and because of the different amounts of impurities especially of salts. While the relative R values are largely independent ofthe water content of the column, the absolute R valuesdepend to a great extent on the amount of water stirred in. It is therefore advisable always to stir the same amount of water into the cellulose suspension, namely the amount at which the optimum sharpness of separation of the vitamin B12 factors can be achieved.

In less complicated cases, single chromatography will be sufficient for the practically complete separation of the individual vitamin B12 factors, because'the cellulose-nbutanol column is exceptionally selective. The vitamin B12 factor III produced in the above-described manner can thereupon be extracted with'water from the solution produced..` After suitable concentration and the addition of, for example, acetone, the Vitamin B12 factor III is crystallized.

The vitamin B12 factor III is characterized in the following manner:

It is ated crystalline neutral substance very similar to` vitamin B12, containing 55.15% l.of carbon, 6.27% of hydrogen, 13.71% of nitrogen, 2.24% of phosphorus and after combustion leaving 11.62% of ash, the ash including Icobalt as a componen-t (all values refer to the anhydrous substance dried at 50 in high vacuum). It

ydiffers from vitamin B12, however, very sharply through its distribution coeiiicient -in certain pairs of solvents,

such as for example in systems consisting of water and mixtures Vof phenols and chlorinated hydrocarbons.

Table I contains a compilation of figures which represent the concentrations of the respective phenols in trichloroethylene, at which the `distribution coeliicient of the vitamin B12 and of vitamin B12 factor III is equal to l. These figures are very characteristic and permit vitamin B12 factor III to be differentiated from the known vitamin B12. y TABLE Iv Distribution of vitamin B12 and vitamin B12 factor III between water and trichloroefhylene-l-phenol compound Concentration oi phenol compound at which the distribution coefeient of the respective vitamin Bn factor in the water] trichloroethylene phenol 'compound is about; 1. Volumetric ratio oi the phases 1:1, Phenol compound pH about7.0

Vitamin B1; Vitamin B11 Factor III a b a b m-chlorophenol. 5. 4 0. 42 8. 7 0. 68 p-chlorophenol 7. 7 0. 6 12. 3 0. 96 3-methyl-4-ehloro-l-hydroxy benzene. 9. 1 0. 64 16.5 1. 16 8.5 0. 90 14.6 1. 55 9. 3 0. 86 16. 8 1.56 m-cresol'. 10. 2 0. 94 17. 7 1. 64 2methyl4-chloro-l-hydroxy benzene 14. 6 1. 02 26. 4 l. 85 2,5-dichlorophenol. 27. 2 1. 67 36. 9 2. 27 2,4-dichlorophenol. 28.7 1. 76 40. 4 1. 48 o-cresol. 16. 8 1. 56 27. 2 2. 52 o-chlorophenol 28.8 2.24 39. 6 3. 08

(a)==G. phenol compound in 100 ce. solution. (b)=Moles of phenol compound in 1 liter of solution.

A further possibility of differentiation is offered by the distribution coefficient in the system n-butanol/water -l-ammonium sulphate, and also the R1 values (see Table II).

1 Determined by means of Whatman 1 paper and water-saturated secondary butanol with the addition of GN", rising.

b G. of ammonium sulphate to 100 cc. of Water.

The novel features which are considered as characteristic for the invention are set forth in particular in the appended claim. The accompanying drawings are however given for the'purpose of better understanding of the distinguishing characteristics of the product of the present invention. In the accompanying drawings:

v Fig. 1 is a graphic representation of the `absorption spectrum of thevitamin B12 factor III in water, theY dottedline referring tov the aquo complex (pH. 4.0), the solid line referring tothe monocyano complex (pH 6.0), and the dashed lirereferring to the'dicyano complex (pH 10.3);

for ,reticulocytes,V erythrocytes and haemoglcbininv the treatment Vof a case of pernicious anaemia with 150 gamma of vitamin B12 factor III.

TABLE n1 containedin the red-coloredy percolate,` from which crystallised Vitamin B12 factor III can advantageously be produced. s

1 U.V. absorption maxima of factor III in water" my 17 mp 17 m1 17 m 17 rn 1 Eiefn. Etalon. l Eicfn. 'L Eieren. 'L Elgin.

Aquocornplex (pH 4) 351 185 500 58. 5 526 61 Monocyanocomplex (pH 6) 295 106 361 201 518 54. 5 550 62. 5 Dleyanocornplex (pH 10.3) 278 83 305 92 368 220 540 65 580 76 As can be seen from Table III, the new vitamin B12 15 EXAMPLE 3 I factor III differs from all hitherto known vitamin B12 factors by the appearance of an absorption band at 295 .m.

The vitamin B12 factor III is electrophoretically neutral in pH'ranges from 1.0 to-9.5 and acidic at pH values over about 9.5. Y

Factor B can be produced from the vitamin B12 factor III, in similar manner as it can be produced from vitamin B12 according to B. J'. Armintage et al., I. Chem. Soc., London, 1953, p. 3849, by the action of concentrated hydrochloric acid at 65 for 5 minutes.

The vitamin B12 factor III has a strong microbiological activity against the E. coli mutant 113-3, L; Zeichimznnii 313 and Oclzromoncls-ma/zrzmensis (Pringheirn).

Thevitamin B12 factor III has a striking and effective action in the therapeutic treatment of pernicious anaemia and other macrocytic anaemias". As an exampleof several examinations, a diagram is given in FigureZ which shows the characteristic curve of the values for reticulocytes, erythrocytes and' haemoglobin in the treatment of a case of pernicious anaemia with V150 'gamma of vitamin B12 factor III. In other cases, a full anti-pernicious effect was achieved by application of only 50 gamma of the same factor. I

The following examples are given for illustrative Vpurposes only, .the scope of the invention not being limited to the specic details of the examples.

EXAMPLE 1 1000 litres of moist digestedV sludge having adry substance coutent of about 10% and a total content of vitamins of the B12 group of 0.5 g. (according to the test with theE. lcoli mutant 113-3) are mixed with a solution of 6 kg. of ferrie chloride and 1 kg. ofsodium bisulphite, and heated for half an hour to 80 at a pH value of 6-7. After sucking olf the sludge with the aid of a vacuum-cell` filter, the practically clear ltrate (about 750 litres) is intimately mixed with 7.5 kg. of charcoal under cold conditions. The adsorbate produced by centrifuging is boiled up with Aartotal `of 75 litres of a mixture ofl parts by volume of n-butanol and 90 parts of water in several portions,and filtered in the'hotstate. to a volume of litres by vacuum distillation. This concentrate now contains 300 mg. of vitamin B12 (according to the test with the E. coli mutant 113-3) including the vitamin B12 factor III.

EXAMPLE 2 50 cc. of an eluate concentrate. produced from digested sludge as in Example 1 is mixed with stirring with 29 g. of ammonium sulphate and 0.5 g. of kieselguhr. This moist product of- 0.3 g. is mixed with 2.8 g. of anhydrous sodium sulphate and groundto a powder. The concentrate of the vitamins ofthe B12 group converted The' resulting eluate is Vconcentrated in this manner into a dry powder is then passed to al small percolator and percolatediwith 15 cc. of a trichloroethylene-phenol mixture having a phenol content ofv 15%.` The remaining percolator material is now brown in color and the @Iltire vitamin B12 activity is 100 cc. of an eluate concentrate of reddish-brow color and produced from a digested sludge, and which' contains 30 gamma lof vitamins of the B12 group per cc. according tothe microbiological test lwith the E. coli mutant 113-3, are brought to a pH of 7 and extracted with 20, 10, and 10 cc. ofa'solution of 20% strength of p-chlorophen'ol in trichloroethylene. The. dark red extracts are combined and washed once with 10 cc. of a solution Vof sodium bicarbonate of 5%- strength and then washed three times more, each time with 10 cc.l of water. i

` EMMPLEA 1 40 cc. of a solution containing 2.9 mg. of vitamins of the B12 group together with Various impurities in a mixture which consists of 20% of p-chlorophenol in trichloroethylene and which originates from a purification process of the vitamins of the B12 group from digested sludge, are mixed with 4 cc. of n-butanol and shaken out four times, each time with 10 cc. of water. The combined aqueous extracts of an intense red color are washed twice, each time with 10 cc. of n-butanol and 3 times each with 15 cc. of ether. An aqueous solution is obtained which has a red color and which contains 2.8 mg. of vitamins of the B12 group (according to the test With the E. coli mutant 113-3). The aqueous solution is thereupon freed in vacuo from organic solvents.

EXAMPLE 5 11 g. of p-chlorophenol and 5 g. of kieselguhr are added to 500cc, of a purified aqueous solution containing 25 gamma per cc. (according to the test with the E. coli mutant 113-3) of Vitamins of B12 group. After shaking for 10 minutes, the red, flaky, readily depositable precipitate is filtered off with the aid of a glass sinter suction filter G3, then mixed with a mixture of 50 cc. of ether and 50 cc. of acetone, ltered off, afterwashed Vwith a little acetone, and freed from acetone in the vacuum exsiccator.

EXAMPLE 6 A concentrate Vof the vitamins of the B12 group produced from 20 litres of digested sludge is introduced with a developer solution consisting of %v of acetone.

and 10% of water and containing 1&0@ mol of sodium cyanide. The water content is gradually increased to 30%. The factor B is practically completely separated from the other components and leaves the column-with a water content of 10-15% in the elution medium in the form of a uniform band. A small amount of cyanocobalamin and finally factor III, which constitutes the bulk of the vitamins of the group, follow. After concentration of the fraction containing the vitamin B12 factor III to a small volume, the factor III is crystallised after addition of acetone.

EXAMPLE 7 .A mXture of vitamin B12 factors, produced from 10 9 A litres of digested sludge and pre-purified, deposited on kieselguhr and dried, is introduced into a column which has been made from cellulose powder, n-butanol and water with the addition of the customary amount of sodium cyanide. The diameter of the column is 2.7 cm. and its height 7 cm. After development with watersaturated n-butanol, containing the usual amount of sodium cyanide, the individual vitamin B12 factors are collected in the eluate in a practically completely separated form. The fraction containing the vitamin B12 factor III is thereupon extracted with water, the aqueous extract is concentrated in vacuo, and the residue is crystallised with acetone.

EXAMPLE 8 1000 litres of moist digested sludge having a dry substance content of 8% and a content of vitamins of the B12 -group amounting to a total of 420 mg. (according to the test with the E. coli mutant 1513-3) are diluted with 1000 litres of water, mixed with 1 kg. of sodium cyanide, brought to a pH of 6.5 with 3 litres of hydrochloric acid of 20% strength, thereupon heated to 80 C., and separated from solid constituents with the aid of a centrifuge. 'Ihe clarified centrifugate is cooled to 20 C. and stirred with 10 kg. of activated charcoal for 30 minutes. The charcoal is separated in the centrifuge and thereupon fractionally eluted with hot ethyl alcohol of 70% strength. 150 litres of eluate are produced, which are concentrated to 10 litres in vacuo. The eluate concentrate is extracted live times, each time with 1000 cc. of a mixture of o-dichlorobenzene and 25% of p-chlorophenol, the combined yextracts are washed twice, each time with 500 cc. of a phosphate buffer of a strength of 2% and a pH of 7, and then three times, each time with 500 cc. of distilled water. The washing liquids are combined and re-extracted with a small volume of o-dichlorobenzene-p-chlorophenol mixture. The combined organic red extracts are mixed with 1000 cc. of n-butanol and extracted several times with small amounts of water, whereby the vitamins of the B12 group pass over into the water.

The aqueous extracts are washed with a little ether and then freed in vacuo from residues of organic solvents. Thus 5 litres of an aqueous solution of pure red color lare obtained, which contain factor III in addition to fother vitamin B12 factors. The aqueous solution is then mixed with 1% of kieselguhr, brought to -a pH of 3 with the aid of hydrochloric acid, and after adding 2.3% of p-chlorophenol is vigorously stirred for 20 minutes, whereupon the vitamins of the B12 group are precipitated on the kieselguhr. The aky red precipitate is sucked off through a layer of lrieselguhr, the lter residue is mixed with a mixture of 1 litre of acetone and 1 litre of ether and stirred for a certain time. The red precipitate freed in `this manner from p-chlorophenol and containing the vitamins of the B12 group is-ltered off, washed with acetone, and dried in vacuo.

T'he dry precipitate is left overnight in an atmosphere containing hydrocyanic acid, thereupon introduced into a cellulose-n-butanol column (diameter 12 cm., height l2 cm.) and chromatographed with water-saturated n-butanol containing 0.005% of hydrocyanic acid. On development of the chromatogram, numerous red or violet bands appear, the lowermost (violet) of which corresponds to factor B. A faint red band (cyanocobalamin) follows, and finally a very thick red band, representing the factor III. After elution of the factor III, there remain -in the middle and at the upper end of the column respectively a few violet bands which correspond to other vitamin B12 factors. The fraction containing the factor III is extracted with water, the aqueous extracts are concentrated in vacuo to a very small volume, and crystallised with acetone. The yield of crystalline product is 150 mg.

Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can by applying current knowledge readily adapt it for various applications without omitting features that, from standpoint of prior art, fairly constitute essential char acter-istics of the generic or specific aspects of this invention and, therefore, such adaptations should and are intended to be comprehended within the meaning and range of equivalence of the following claim.

What is claimed as new and desired to be Secured by Letters Patent is:

As a new composition of matter, vitamin B12 factor III having the structural formula shown in Fig. 3 of Y the drawing.

Lightfoot et al. Apr. 2, 1957 Bernhauser et al. Oct. 8. 1957 

